CN105121653A - Stabilized formulation for luminescent detection of luciferase and nucleoside phosphates - Google Patents

Stabilized formulation for luminescent detection of luciferase and nucleoside phosphates Download PDF

Info

Publication number
CN105121653A
CN105121653A CN201480010140.5A CN201480010140A CN105121653A CN 105121653 A CN105121653 A CN 105121653A CN 201480010140 A CN201480010140 A CN 201480010140A CN 105121653 A CN105121653 A CN 105121653A
Authority
CN
China
Prior art keywords
fluorescein
composition
luciferase
atp
time period
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201480010140.5A
Other languages
Chinese (zh)
Other versions
CN105121653B (en
Inventor
迈克尔·P·瓦利
詹姆士·J·卡利
布洛克·宾考斯基
克里斯多弗·托德·艾格斯
基思·V·伍德
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pu Luomaige Co
Promega Corp
Original Assignee
Pu Luomaige Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pu Luomaige Co filed Critical Pu Luomaige Co
Priority to CN201911407470.9A priority Critical patent/CN111004835B/en
Priority to CN202311707362.XA priority patent/CN117701675A/en
Publication of CN105121653A publication Critical patent/CN105121653A/en
Application granted granted Critical
Publication of CN105121653B publication Critical patent/CN105121653B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Methods, kits and compositions containing a mixture of D-luciferin and L-luciferin for light generation with luciferase are disclosed that have improved stability when stored over time. The mixture of D-luciferin and L-luciferin can be used to detect the presence or amount of ATP or of luciferase in a sample.

Description

For the stabilization formulations of the luminous detection of luciferase and nucleoside phosphorylase
The cross reference of related application
This application claims the U.S. Provisional Application No.61/767 submitted on February 22nd, 2013, the U.S. Provisional Application No.61/783 that on March 14th, 875 and 2013 submits to, the right of priority of 726, these two provisional application all by way of reference entirety are incorporated to herein.
Background technology
Fluorescein is racemic compound, in the solution, its by D-fluorescein racemize to L-fluorescein or by L-fluorescein racemize to D-fluorescein.D-fluorescein can be used as the substrate of luciferase and produce light, and L-fluorescein is inhibition to a great extent and cause luminous minimizing.Due to the change of the operability of D-fluorescein substrate and the restraining effect of reduction and L-fluorescein, in storage process, D-fluorescein has problems to the slow racemize of L-fluorescein for utilizing the mensuration of uciferase activity.
Technical field
The disclosure relates to composition, method and test kit for measuring enzyme and meta-bolites.
Summary of the invention
In certain embodiments, provide the composition comprising luciferase, L-fluorescein and D-fluorescein, wherein said composition is substantially free of ATP.In certain embodiments, provide the composition comprising ATP, L-fluorescein and D-fluorescein, wherein said composition is substantially free of luciferase.
In some embodiments, the composition comprising D-fluorescein, L-fluorescein and ATP is provided.
In certain embodiments, provide the composition comprising luciferase, L-fluorescein and D-fluorescein and ATP, the concentration of wherein said L-fluorescein exceedes the concentration of described D-fluorescein.
In some embodiments, provide the test kit comprising composition, described composition comprises luciferase, L-fluorescein and D-fluorescein, and described composition is substantially free of ATP and packs in a reservoir.Described test kit can comprise at least one additional component, such as one or more washing composition, such as dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, potassiumphosphate, defoamer, buffer reagent, salt and sequestrant, such as ethylenediamine tetraacetic acid (EDTA) (EDTA).
In some embodiments, provide the test kit comprising composition, described composition comprises D-fluorescein, L-fluorescein and ATP, and the concentration of wherein said L-fluorescein exceedes the concentration of described D-fluorescein.Described test kit can comprise at least one additional component, such as one or more washing composition, such as dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, potassiumphosphate, defoamer, buffer reagent, salt and sequestrant, such as ethylenediamine tetraacetic acid (EDTA) (EDTA).
In some embodiments, provide the test kit comprising composition, described composition comprises L-fluorescein, D-fluorescein and ATP, and described composition is substantially free of luciferase and packs in a reservoir.Described test kit can comprise at least one additional component, such as buffer reagent, divalent cation chelators, magnesium salts, ionic or non-ionic detergent, containing mercaptan compound, as coenzyme A or DTT, one or more sulfur-bearing reductive agents or its combination.
In some embodiments, provide the method for existence for ATP in working sample or amount, wherein make to comprise the composition of luciferase, L-fluorescein and D-fluorescein and sample contacts and detect and/or measure the luminous intensity that produces with the existence of ATP in working sample or amount.
In some embodiments, provide for the method for the existence of luciferase in working sample or amount, test kit or composition, wherein make the composition and the sample contacts that comprise D-fluorescein, L-fluorescein and ATP, described sample can contain the cell of expressing luciferase.Then detect and/or measure the luminous intensity of generation with the existence of luciferase in working sample or amount.The sample of the cell containing expressing luciferase can be the cell extract of the cell of cracking, unprocessed cell extract or clarification.
In some embodiments, provide the method for the uciferase activity for detecting the cell from expressing luciferase, wherein luciferase is at cells, and make described cell and comprise the mixture of L-fluorescein with D-fluorescein and contact, described in described mixture, the ratio of L-fluorescein and described D-fluorescein is at least about 5:95 to about 75:25.In some embodiments, described mixture comprises L-fluorescein and D-fluorescein, and the ratio of described L-fluorescein and described D-fluorescein is at least about 5:95 to about 55:45, or at least about 5:95 to about 50:50.Then detect and/or measure the luminous intensity of generation to measure existence or the amount of luciferase in cell.
In some embodiments, provide for detecting or the method in nucleoside phosphorylase such as ATP or ATP source in quantify cellular or other samples, wherein make described cell or sample and comprise the mixture of L-fluorescein with D-fluorescein and contact with luciferase, described in described mixture, the ratio of L-fluorescein and described D-fluorescein is at least about 0.5:1 to about 2:1.Then detect and/or measure the luminous intensity of generation to measure existence or the amount of nucleoside phosphorylase in cell.
In some embodiments, the method for carrying out luciferase reaction is provided.The mixture comprising L-fluorescein and D-fluorescein is stored certain hour section at the temperature of about 0 DEG C to about 35 DEG C.When using described mixture in measuring at uciferase activity, measure light output, i.e. luminous intensity.Allow described mixture and contain or expressing luciferase, the sample in ATP or ATP source or sample segment contact, measure thing with forming reactions.Then detection reaction measures the luminous intensity produced in thing.The reaction assay produce third contact of a total solar or lunar eclipse containing mixture having stored certain hour section exports, in certain embodiments, described light output be the light output of (time t=0) comparable reaction assay thing containing described mixture when the storage time, section started at least about 50% or at least about 90%.In certain embodiments, storage time section may be at least about 30 minutes, at least about 1 hour, at least about 4 hours or longer.
In some embodiments, the method for preparing the component for luciferase reaction is provided.D-fluorescein and L-fluorescein are merged to form mixture, and described mixture stores certain hour section at the temperature of about 0 DEG C to about 35 DEG C, according to appointment the time period of 1,2,3 or 4 hour or 1 week or 2 weeks.When using in measuring at uciferase activity, described mixture allows to produce light output, i.e. luminous intensity, thus the light output produced after the described time period be light output when the described time period starts at least about 50% or at least about 90%.
The disclosure relates to the composition comprising luciferase, L-fluorescein and D-fluorescein.Described composition is substantially free of ATP.Described composition can comprise at least about 0.5:1 to the L-fluorescein of about 2:1 and the ratio of D-fluorescein.Described composition can comprise at least about 5:95 to the L-fluorescein of about 55:45 and the ratio of D-fluorescein.Described composition can have at least about 5 and be less than the pH of about 9.Described luciferase can be beetle luciferase, as Photinus pyralis LUC or Pleonomus luciferase.D-fluorescein can be D-5 ' fluorine fluorescein and L-fluorescein is L-5 ' fluorine fluorescein.Described composition can also comprise at least one and be selected from following additional component: buffer reagent, protein stabilizing agent, magnesium ion, metal chelator, washing composition, defoamer, inorganic phosphate, pyrophosphate salt, AMP, coenzyme A, reductive agent or its combination.Described composition can comprise magnesium sulfate and anti-form-1,2-cyclohexanediaminetetraacetic acid (CDTA).Described composition can also comprise at least one and be selected from following additional component: 1,4-piperazine two ethyl sulfonic acid, 1,2-diamino-cyclohexane tetraacethyl, Tergitol washing composition, Mazu defoamer, magnesium sulfate, ATP, coenzyme A, pyrophosphate salt, DTT, sulphite and thiosulphate.Described composition can also comprise at least one and be selected from following component: Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate, ethylenediamine tetraacetic acid (EDTA) (EDTA) and AMP.Described composition can comprise at least two kinds in described additional component.Described composition can comprise at least three kinds in described additional component.Described composition can comprise at least four kinds in described additional component.Described composition can comprise at least five kinds in described additional component.Described composition can also comprise magnesium ion, citric acid buffer agent, MES buffer reagent, Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate and ethylenediamine tetraacetic acid (EDTA) (EDTA).Described composition can also comprise citric acid buffer agent, HEPES buffer reagent, magnesium ion, ethylenediamine tetraacetic acid (EDTA) (EDTA), potassiumphosphate, defoamer, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane and AMP.Compared with not comprising the composition of L-fluorescein, when storing at the temperature between about 0 DEG C to about 35 DEG C, described composition can have storage time and/or the stability of increase.
The disclosure relates to the composition comprising D-fluorescein, L-fluorescein and ATP.Described composition is substantially free of luciferase.Described composition can comprise at least about 0.5:1 to the L-fluorescein of about 2:1 and the ratio of D-fluorescein.Described composition can comprise at least about 5:95 to the L-fluorescein of about 55:45 and the ratio of D-fluorescein.Described composition can have at least about 5 and be less than the pH of about 9.Described luciferase can be beetle luciferase, as Photinus pyralis LUC or Pleonomus luciferase.D-fluorescein can be D-5 ' fluorine fluorescein and L-fluorescein is L-5 ' fluorine fluorescein.Described composition can also comprise at least one and be selected from following additional component: buffer reagent, protein stabilizing agent, magnesium ion, metal chelator, washing composition, defoamer, inorganic phosphate, pyrophosphate salt, AMP, coenzyme A, reductive agent or its combination.Described composition can comprise magnesium sulfate and anti-form-1,2-cyclohexanediaminetetraacetic acid (CDTA).Described composition also comprises at least one and is selected from following additional component: 1,4-piperazine two ethyl sulfonic acid, 1,2-diamino-cyclohexane tetraacethyl, Tergitol washing composition, Mazu defoamer, magnesium sulfate, ATP, coenzyme A, pyrophosphate salt, DTT, sulphite and thiosulphate.Described composition also comprises at least one and is selected from following component: Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate, ethylenediamine tetraacetic acid (EDTA) (EDTA) and AMP.Described composition can comprise at least two kinds in described additional component.Described composition can comprise at least three kinds in described additional component.Described composition can comprise at least four kinds in described additional component.Described composition can comprise at least five kinds in described additional component.Described composition can also comprise magnesium ion, citric acid buffer agent, MES buffer reagent, Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate and ethylenediamine tetraacetic acid (EDTA) (EDTA).Described composition can also comprise citric acid buffer agent, HEPES buffer reagent, magnesium ion, ethylenediamine tetraacetic acid (EDTA) (EDTA), potassiumphosphate, defoamer, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane and AMP.Compared with not comprising the composition of L-fluorescein, when storing at the temperature between about 0 DEG C to about 35 DEG C, described composition can have storage time and/or the stability of increase.
The disclosure relates to the composition comprising D-fluorescein, L-fluorescein and ATP, and the concentration of wherein said L-fluorescein exceedes the concentration of described D-fluorescein.Described composition can have at least about 5 and be less than the pH of about 9.Described luciferase can be beetle luciferase, as Photinus pyralis LUC or Pleonomus luciferase.D-fluorescein can be D-5 ' fluorine fluorescein and L-fluorescein is L-5 ' fluorine fluorescein.Described composition can also comprise at least one and be selected from following additional component: buffer reagent, protein stabilizing agent, magnesium ion, metal chelator, washing composition, defoamer, inorganic phosphate, pyrophosphate salt, AMP, coenzyme A, reductive agent or its combination.Described composition can comprise magnesium sulfate and anti-form-1,2-cyclohexanediaminetetraacetic acid (CDTA).Described composition can also comprise at least one and be selected from following additional component: 1,4-piperazine two ethyl sulfonic acid, 1,2-diamino-cyclohexane tetraacethyl, Tergitol washing composition, Mazu defoamer, magnesium sulfate, ATP, coenzyme A, pyrophosphate salt, DTT, sulphite and thiosulphate.Described composition can also comprise at least one and be selected from following component: Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate, ethylenediamine tetraacetic acid (EDTA) (EDTA) and AMP.Described composition can comprise at least two kinds in described additional component.Described composition can comprise at least three kinds in described additional component.Described composition can comprise at least four kinds in described additional component.Described composition can comprise at least five kinds in described additional component.Described composition can also comprise magnesium ion, citric acid buffer agent, MES buffer reagent, Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate and ethylenediamine tetraacetic acid (EDTA) (EDTA).Described composition can comprise citric acid buffer agent, HEPES buffer reagent, magnesium ion, ethylenediamine tetraacetic acid (EDTA) (EDTA), potassiumphosphate, defoamer, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane and AMP.Compared with not comprising the composition of L-fluorescein, when storing at the temperature between about 0 DEG C to about 35 DEG C, described composition can have storage time and/or the stability of increase.
The disclosure relates to and has the storage time of increase and/or the composition of stability.Described composition can comprise luciferase, L-fluorescein and D-fluorescein, and wherein said composition is substantially free of ATP.Described luciferase can be beetle luciferase.Described beetle luciferase can be Photinus pyralis LUC or Pleonomus luciferase.Described composition can comprise at least about 0.5:1 to the L-fluorescein of about 2:1 and the ratio of D-fluorescein.Described composition can comprise at least about 5:95 to the L-fluorescein of about 55:45 and the ratio of D-fluorescein.Compared with not comprising the composition of L-fluorescein, when storing at the temperature of about 0 DEG C to about 35 DEG C, described reaction mixture can have storage time and/or the stability of increase.Described composition can have at least about 5 and be less than the pH of about 9.D-fluorescein can be D-5 ' fluorine fluorescein, and L-fluorescein is L-5 ' fluorine fluorescein.Described composition can also comprise at least one and be selected from following additional component: buffer reagent, protein stabilizing agent, magnesium ion, metal chelator, washing composition, defoamer, inorganic phosphate, pyrophosphate salt, AMP, coenzyme A, reductive agent or its combination.Described composition can comprise magnesium sulfate and anti-form-1,2-cyclohexanediaminetetraacetic acid (CDTA).Described composition can also comprise at least one and be selected from following additional component: 1,4-piperazine two ethyl sulfonic acid, 1,2-diamino-cyclohexane tetraacethyl, Tergitol washing composition, Mazu defoamer, magnesium sulfate, ATP, coenzyme A, pyrophosphate salt, DTT, sulphite and thiosulphate.Described composition can also comprise at least one and be selected from following component: Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate, ethylenediamine tetraacetic acid (EDTA) (EDTA) and AMP.Described composition can comprise at least two kinds in described additional component.Described composition can comprise at least three kinds in described additional component.Described composition can comprise at least four kinds in described additional component.Described composition can comprise at least five kinds in described additional component.Described composition can also comprise magnesium ion, citric acid buffer agent, MES buffer reagent, Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate and ethylenediamine tetraacetic acid (EDTA) (EDTA).Described composition can comprise citric acid buffer agent, HEPES buffer reagent, magnesium ion, ethylenediamine tetraacetic acid (EDTA) (EDTA), potassiumphosphate, defoamer, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane and AMP.
The disclosure relates to and has the storage time of increase and/or the composition of stability.Described composition comprises D-fluorescein, L-fluorescein and ATP, and wherein said composition is substantially free of luciferase.Described composition can comprise at least about 0.5:1 to the L-fluorescein of about 2:1 and the ratio of D-fluorescein.Described composition can comprise at least about 5:95 to the L-fluorescein of about 55:45 and the ratio of D-fluorescein.Compared with not comprising the composition of L-fluorescein, when storing at the temperature of about 0 DEG C to about 35 DEG C, described reaction mixture can have storage time and/or the stability of increase.Described composition can have at least about 5 and be less than the pH of about 9.D-fluorescein can be D-5 ' fluorine fluorescein, and L-fluorescein is L-5 ' fluorine fluorescein.Described composition can also comprise at least one and be selected from following additional component: buffer reagent, protein stabilizing agent, magnesium ion, metal chelator, washing composition, defoamer, inorganic phosphate, pyrophosphate salt, AMP, coenzyme A, reductive agent or its combination.Described composition can comprise magnesium sulfate and anti-form-1,2-cyclohexanediaminetetraacetic acid (CDTA).Described composition can also comprise at least one and be selected from following additional component: 1,4-piperazine two ethyl sulfonic acid, 1,2-diamino-cyclohexane tetraacethyl, Tergitol washing composition, Mazu defoamer, magnesium sulfate, ATP, coenzyme A, pyrophosphate salt, DTT, sulphite and thiosulphate.Described composition can also comprise at least one and be selected from following component: Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate, ethylenediamine tetraacetic acid (EDTA) (EDTA) and AMP.Described composition can comprise at least two kinds in described additional component.Described composition can comprise at least three kinds in described additional component.Described composition can comprise at least four kinds in described additional component.Described composition can comprise at least five kinds in described additional component.Described composition can also comprise magnesium ion, citric acid buffer agent, MES buffer reagent, Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate and ethylenediamine tetraacetic acid (EDTA) (EDTA).Described composition can comprise citric acid buffer agent, HEPES buffer reagent, magnesium ion, ethylenediamine tetraacetic acid (EDTA) (EDTA), potassiumphosphate, defoamer, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane and AMP.
The disclosure relates to and has the storage time of increase and/or the composition of stability.Described composition comprises D-fluorescein, L-fluorescein and ATP, and the concentration of wherein said L-fluorescein exceedes the concentration of described D-fluorescein.Compared with not comprising the composition of L-fluorescein, when storing at the temperature of about 0 DEG C to about 35 DEG C, described reaction mixture can have storage time and/or the stability of increase.Described composition can have at least about 5 and be less than the pH of about 9.D-fluorescein can be D-5 ' fluorine fluorescein, and L-fluorescein is L-5 ' fluorine fluorescein.Described composition can also comprise at least one and be selected from following additional component: buffer reagent, protein stabilizing agent, magnesium ion, metal chelator, washing composition, defoamer, inorganic phosphate, pyrophosphate salt, AMP, coenzyme A, reductive agent or its combination.Described composition can comprise magnesium sulfate and anti-form-1,2-cyclohexanediaminetetraacetic acid (CDTA).Described composition can also comprise at least one and be selected from following additional component: 1,4-piperazine two ethyl sulfonic acid, 1,2-diamino-cyclohexane tetraacethyl, Tergitol washing composition, Mazu defoamer, magnesium sulfate, ATP, coenzyme A, pyrophosphate salt, DTT, sulphite and thiosulphate.Described composition can also comprise at least one and be selected from following component: Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate, ethylenediamine tetraacetic acid (EDTA) (EDTA) and AMP.Described composition can comprise at least two kinds in described additional component.Described composition can comprise at least three kinds in described additional component.Described composition can comprise at least four kinds in described additional component.Described composition can comprise at least five kinds in described additional component.Described composition can also comprise magnesium ion, citric acid buffer agent, MES buffer reagent, Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate and ethylenediamine tetraacetic acid (EDTA) (EDTA).Described composition can also comprise citric acid buffer agent, HEPES buffer reagent, magnesium ion, ethylenediamine tetraacetic acid (EDTA) (EDTA), potassiumphosphate, defoamer, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane and AMP.
The disclosure relates to the test kit comprising the composition as above be packaged at least one container.
The disclosure relates to test kit, described test kit comprises and is packaged in composition as above in the first container and second container, and described second container comprises at least one and is selected from following component: citric acid buffer agent, MES buffer reagent, Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate, ethylenediamine tetraacetic acid (EDTA) (EDTA) and AMP.Compared with not comprising the composition of L-fluorescein, when storing at the temperature between about 0 DEG C to about 35 DEG C, described composition can have storage time and/or the stability of increase.
The disclosure relates to the method for existence for ATP in working sample or amount, and it comprises makes composition as above and sample contacts, and detects the luminous intensity produced, thus the existence of ATP or amount in working sample.Described sample can comprise complete cell.Described sample comprises the cell lysate of unprocessed cell lysate or clarification.Described cell can be prokaryotic cell prokaryocyte or eukaryotic cell.Described sample can comprise purified enzyme, and described enzyme produces or uses ATP.Compared with not comprising the composition of L-fluorescein, when storing at the temperature between about 0 DEG C to about 35 DEG C, described composition can have storage time and/or the stability of increase.
The disclosure relates to the method for uciferase activity in the cell for detecting expressing luciferase, and described method is included in cells luciferase; Make described luciferase, described cell or its combination and comprise the mixture of L-fluorescein with D-fluorescein and contact and detect luminous intensity, described in described mixture, the ratio of L-fluorescein and described D-fluorescein is that (i) is at least about 5:95 to about 75:25, (ii) at least about 5:95 to about 50:50, (iii) at least about 5:95 to about 25:75 or (iv) at least about 5:95 to about 20:80.Described cell can be cleaved, and the cell comprising the cracking of luciferase can contact with described mixture.The cell of cracking can be made to clarify to form the cell extract comprising the clarification of luciferase, and the cell extract of described clarification is contacted with described mixture.Described cell can be eukaryotic cell, as mammalian cell.Described mixture may increase the transformation period of luminous signal.Cell described in luciferase-encoding sequences transient transfection can be used.Described mixture can have at least about 6.5 to the pH being less than about 7.8, or at least about 6.6 at least about 6.8 pH.D-fluorescein can be D-5 ' fluorine fluorescein and L-fluorescein is L-5 ' fluorine fluorescein.Compared with not comprising the composition of L-fluorescein, when storing at the temperature between about 0 DEG C to about 35 DEG C, described composition can have storage time and/or the stability of increase.Described mixture can have the pH at least about 5 to about 9.
The disclosure relates to the method for carrying out luciferase reaction.Described method comprises: the time period that (a) will store at the temperature of the mixture comprising L-fluorescein and D-fluorescein between about 0 DEG C to about 35 DEG C at least about 4 hours, wherein when merging with sample in the mensuration thing comprising luciferase and ATP, the mixture of described storage allows to produce light output, and the light output after the wherein said time period be light output when starting the described time period at least about 50%; And (b) is after step (a), allow the mixture of described storage and (i) sample and (ii) ATP or ATP source, luciferase or its combine to merge and measure with forming reactions the luminous intensity that thing and detection produce.The described time period can be at least about 12 hours, and described temperature can be about 0 DEG C to about 6 DEG C or about 20 DEG C to about 35 DEG C.Described sample can comprise luciferase, and described mixture comprises ATP.Described sample can comprise ATP, and described mixture comprises luciferase.Light output after the described time period be light output when starting the described time period at least about 75%.Described mixture can be substantially free of ATP or luciferase.Described temperature is about 20 DEG C to about 35 DEG C.Described temperature can be about 0 DEG C to about 6 DEG C, and the described time period is at least about 2 weeks.The described time period can be at least about 12 weeks.The described time period can be at least about 26 weeks.Light output after the described time period can for the described time period start time light output at least about 90%.Light output after the described time period can for the described time period start time light output at least about 50%.After described method can also be included in step (b), by the mixture of described storage and (i) luciferase and, (ii) ATP or ATP source or (iii) its combine and merge to form the second mixture, and detect the luminous intensity produced in described second mixture.Described mixture can have the pH at least about 5 to about 9.Described mixture can comprise at least about 5:95 to the L-fluorescein of about 75:25 and the ratio of D-fluorescein.
The disclosure relates to the method for preparing the component for luciferase reaction.Described method comprises: D-fluorescein and L-fluorescein merge to form mixture by (a), and described mixture is stored the time period at least about 2 days by (b) at the temperature of about 0 DEG C to about 35 DEG C.When merging with sample in the mensuration thing comprising luciferase, described mixture allows to produce light output, and the light output after the wherein said time period be light output when starting the described time period at least about 50%.Described mixture can be substantially free of ATP or luciferase.Described temperature can be about 20 DEG C to about 35 DEG C.Described temperature can be about 0 DEG C to about 6 DEG C, and the described time period is at least about 2 weeks.The described time period can be at least about 12 weeks.The described time period can be at least about 26 weeks.Light output after the described time period can for the described time period start time light output at least about 90%.Light output after the described time period can for the described time period start time light output at least about 50%.After described method can also be included in step (b), by the mixture of described storage and (i) luciferase, (ii) ATP or ATP source or (iii) its combine and merge to form the second mixture, and detect the luminous intensity produced in described second mixture.Described mixture can have the pH at least about 5 to about 9.Described composition can comprise at least about 5:95 to the L-fluorescein of about 75:25 and the ratio of D-fluorescein.
The disclosure relates to the method for the transformation period for increasing luminous signal.Described method comprises: the sample that (a) makes to comprise luciferase with comprise D-fluorescein, L-fluorescein and to contact with the mixture of ATP and measure thing with forming reactions; And (b) detects luminous intensity; Wherein with comprise D-fluorescein and compared with the comparable reaction assay thing being substantially free of L-fluorescein, the transformation period of luminous signal increases.The transformation period of described luminous signal can increase at least about 25%.The transformation period of described luminous signal can increase at least about 5 times.Described sample can comprise the cell of expressing luciferase.Described cell can be cleaved, and described sample can comprise the cell of cracking, and the cell of described cracking comprises luciferase.Described sample can comprise the cell extract of clarification, and described cell extract comprises luciferase.Described cell can be prokaryotic cell prokaryocyte or eukaryotic cell.D-fluorescein can be D-5 ' fluorine fluorescein, and L-fluorescein is L-5 ' fluorine fluorescein, and wherein comparable reaction assay thing comprises D-5 ' fluorine fluorescein and is substantially free of L-5 ' fluorine fluorescein.
The disclosure relates to the method for carrying out luciferase reaction.Described method comprises: (a) by composition and sample and ATP or ATP source, luciferase or its combine to merge and measure thing with forming reactions, described composition comprises L-fluorescein and D-fluorescein; And (b) detects the luminous intensity produced, wherein compared with not comprising the composition of L-fluorescein, if described composition stores the time period at least about 4 hours at the temperature of about 0 DEG C to about 35 DEG C, then described composition has the stability of increase.When merging with sample in the mensuration thing comprising luciferase and ATP, the composition of described storage allows to produce light output, and the light output after the wherein said time period be light output when starting the described time period at least about 50%.The stability of described increase can comprise the storage time of increase and/or the signal stabilization of increase.The described time period can be at least about 12 hours, and described temperature is about 0 DEG C to about 6 DEG C or about 20 DEG C to about 35 DEG C.Described sample can comprise luciferase, and described composition can comprise ATP.Described sample can comprise ATP, and described mixture comprises luciferase.Light output after the described time period can for the described time period start time light output at least about 75%.Described composition can be substantially free of ATP or luciferase.Described temperature can be about 20 DEG C to about 35 DEG C.Described temperature can be about 0 DEG C to about 6 DEG C, and the described time period is at least about 2 weeks.The described time period can be at least about 12 weeks.The described time period can be at least about 26 weeks.Light output after the described time period can for the described time period start time light output at least about 90%.Light output after the described time period can for the described time period start time light output at least about 50%.After described method is also included in step (b), by described composition and (i) luciferase, (ii) ATP or ATP source or (iii) its combine and merge to form mixture, and detect the luminous intensity produced in described mixture.Described composition can have the pH at least about 5 to about 9.Described composition can comprise at least about 5:95 to the L-fluorescein of about 75:25 and the ratio of D-fluorescein.
The disclosure relates to the method for preparing the component for luciferase reaction, described method comprises and merges D-fluorescein and L-fluorescein to form composition, wherein compared with not comprising the composition of L-fluorescein, if described composition stores the time period of at least 4 hours at the temperature of about 0 DEG C to about 35 DEG C, then described composition has the stability of increase, wherein when merging with sample in the mensuration thing comprising luciferase and ATP, the composition of described storage allows to produce light output, and the light output after the wherein said time period be light output when starting the described time period at least about 50%.The stability of described increase can comprise the storage time of increase and/or the signal stabilization of increase.Described composition can be substantially free of ATP or luciferase.Described temperature can be about 20 DEG C to about 35 DEG C.Described temperature can be about 0 DEG C to about 6 DEG C, and the described time period is at least about 2 weeks.The described time period can be at least about 12 weeks.The described time period can be at least about 26 weeks.Light output after the described time period can for the described time period start time light output at least about 90%.Light output after the described time period can for the described time period start time light output at least about 50%.After described method is also included in step (b), by described composition and (i) luciferase, (ii) ATP or ATP source or (iii) its combine and merge to form mixture, and detect the luminous intensity produced in described mixture.Described composition can have the pH at least about 5 to about 9.Described composition can comprise at least about 5:95 to the L-fluorescein of about 75:25 and the ratio of D-fluorescein.
By considering detailed description book and accompanying drawing, other aspects of the present invention will become apparent.
Accompanying drawing explanation
Fig. 1 is the figure describing the luminous intensity surveyed by the mensuration quality testing containing L-fluorescein and D-fluorescein after fluorescein mixture stores the different time at different temperatures.
Fig. 2 is for describing the figure of the luminous intensity surveyed by the mensuration quality testing containing D-fluorescein or L-fluorescein and D-fluorescein after fluorescein stores the different time.
Fig. 3 is for describing the figure of the luminous intensity surveyed by the mensuration quality testing containing D-fluorescein or L-fluorescein and D-fluorescein after fluorescein stores the different time.
Fig. 4 shows and describes luciferase reporter gene and measure the figure of D-/L-fluorescein (5 '-fluorine fluorescein) ratios different in reagent on luminous intensity (4A) and the impact of signal transformation period (4B).
Fig. 5 describes different D-/L-fluorescein (5 '-fluorine fluorescein) ratios measures the impact of the stability of reagent figure on luciferase reporter gene.
Describe in detail
L-fluorescein between the shelf lives in liquid preparation is to the mixture of D-and the L-enantiomer of the racemization generation fluorescein of D-fluorescein, and wherein D-fluorescein can be used as by the substrate of luciferase to produce light.The unpredictability that can be used for the amount of the D-fluorescein of luciferase is debatable to depending on the mensuration measuring uciferase activity, if meta-bolites in measure sample is as the mensuration of the amount of ATP.In mensuration, the existence of L-fluorescein also may be debatable, because it serves as the inhibitor of luciferase.
In some embodiments, the composition that D-fluorescein and the L-fluorescein provided as a mixture is provided is disclosed.In some embodiments, described composition can use to measure the enzyme such as kinase whose activity using ATP together with luciferase; To measure amount or the concentration of ATP; With the specific meta-bolites by producing in the middle of ATP in measure sample; To provide through the stably measured of certain hour section or its combination.In some embodiments, described composition may be used for detecting or measuring by the amount of the luciferase of cell expressing.Surprisingly, the composition of the mixture containing D-fluorescein and L-fluorescein is compared to the composition containing substantially pure D-fluorescein or comprise D-fluorescein and be substantially free of the stability of the composition exhibiting excellence of L-fluorescein (as containing micro-L-fluorescein), and wherein mensuration degradation is minimum.Such as, when L-fluorescein with exist total fluorescein at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70% or when existing at least about 75% or more, or when the amount of L-fluorescein exceedes the amount of D-fluorescein of existence, excellent stability may be there is.Can include but not limited to by producing the meta-bolites measured in the middle of ATP, nucleoside diphosphate is as adenosine diphosphate (ADP) (ADP), guanosine diphosphate(GDP) (GDP), uridine diphosphate (UDP) (UDP) and adenosine monophosphate (AMP).In certain embodiments, the composition comprising the mixture of D-fluorescein and L-fluorescein optionally comprises luciferase, and can optionally be substantially free of ATP, other ribonucleoside triphosphote, nucleoside diphosphate, Nucleotide monophosphates or its combination.In other embodiments, the mixture comprising D-fluorescein and L-fluorescein such as wherein the concentration of L-fluorescein optionally can comprise ATP more than the composition of the mixture of the concentration of D-fluorescein.
D-fluorescein comprises compound (D-(-)-2-(6 '-hydroxyl-2 '-benzothiazolyl)-thiazoline-4-carboxylic acid), and L-fluorescein comprises compound (L-(-)-2-(6 '-hydroxyl-2 '-benzothiazolyl)-thiazoline-4-carboxylic acid) and their salt form.As used herein, no matter be D-or L-isomeric form, fluorescein comprises salt form, as the form of sylvite, sodium salt or other basic metal or alkaline earth salt, free acid and fluorescein derivative and their salt, as chlorine fluorescein and fluorine fluorescein, such as 5 '-fluorine fluorescein, 7 '-fluorine fluorescein and 5 '-chlorine fluorescein and 7 '-chlorine fluorescein and in U.S. published application 2009-0075309 disclosed those, whole disclosures of described U.S. published application are incorporated to herein by reference.
D-fluorescein and L-fluorescein (D-fluorescein: L-fluorescein) can with at least about 0.25:1, at least about 0.3:1, at least about 0.4:1, at least about 0.5:1, at least about 0.6:1, at least about 0.7:1, at least about 0.8:1, at least about 0.9:1, about 4:1 is less than at least about 1:1, be less than about 3.75:1, be less than about 3.5:1, be less than about 3.25:1, be less than about 3:1, be less than about 2.75:1, be less than about 2.5:1, be less than about 2.25:1, be less than about 2:1, be less than 1.9:1, be less than about 1.8:1, be less than about 1.7:1, be less than about 1.6:1, be less than about 1.5:1, be less than about 1.4:1, be less than about 1.3:1, be less than about 1.25:1, be less than about 1.2:1 or be less than the ratio being about less than about 1.1:1 and exist.
D-fluorescein and L-fluorescein (D-fluorescein: L-fluorescein) can with at least about 0.1:99.9, at least about 1:99, at least about 2:98, at least about 3:97, at least about 4:96, at least about 5:95, at least about 10:90, at least about 15:85, at least about 20:80, at least about 25:75, at least about 30:70, at least about 35:65, at least about 40:60, at least about 45:55, at least about 49:51, or be less than about 99:0.1 at least about 50:50, be less than about 99:1, be less than about 98:2, be less than about 97:3, be less than about 96:4, be less than about 95:5, be less than about 90:10, be less than about 85:15, be less than about 80:20, be less than about 75:25, be less than about 70:30, be less than about 65:35, be less than about 60:40, be less than about 55:45, be less than about 51:49 or be less than about 50:50 ratio exist.
The mixture comprising D-fluorescein, L-fluorescein and optionally luciferase can be substantially free of or get rid of the ribonucleoside triphosphote of any amount, nucleoside diphosphate or their arbitrary combination.Such as, the mixture comprising D-fluorescein, L-fluorescein and optionally luciferase can be substantially free of or get rid of ATP, GTP, CTP, m5UTP, UTP or their arbitrary combination of any amount.The mixture comprising D-fluorescein, L-fluorescein and optionally luciferase can be substantially free of or get rid of ADP, GDP, CDP, m5UDP, UDP or their arbitrary combination of any amount.The mixture comprising D-fluorescein, L-fluorescein and optionally luciferase can comprise and be less than about 50 μm of ATP, is less than about 10 μm of ATP, is less than about 5 μm of ATP, is less than about 1 μm of ATP, is less than about 0.5 μm of ATP, is less than about 0.1 μm of ATP, is less than about 0.05 μm of ATP, is less than about 0.01 μm of ATP, is less than about 0.005 μm of ATP or is less than about 0.001 μm of ATP or is less than about 0.0001 μm of ATP or other ribonucleoside triphosphote as herein described.
In some embodiments, the mixture comprising D-fluorescein, L-fluorescein and optionally luciferase can containing at least 0.005 μM of ATP, at least about 0.01ATP, at least about 0.05 μM of ATP, at least about 0.1 μM of ATP, at least about 0.5 μM of ATP, at least about 1 μM of ATP, at least about 5 μMs of ATP, be less than about 10mMATP at least about 10 μMs of μMs of ATP, at least about 50 μMs of ATP, be less than about 5mMATP, be less than about 4mMATP, be less than about 3mMATP, be less than about 2mMATP, be less than about 1mMATP, be less than about 0.5mMATP.Such as, such mixture can comprise and be greater than 1:1, is greater than 1.1:1, is greater than 1.2:1, is greater than 1.3:1, is greater than 1.4:1, is greater than 1.5:1, is greater than 1.6:1, is greater than 1.7:1, is greater than 1.8:1, is greater than 1.9:1, is greater than 2:1, is greater than 2.25:1, is greater than 2.5:1, is greater than 2.75:1 or is greater than the L-fluorescein of 3:1: the ratio of D-fluorescein.
The mixture comprising D-fluorescein and L-fluorescein optionally can consist essentially of the ribonucleoside triphosphote of any amount, such as ATP, GTP, CTP, nucleoside diphosphate, such as ADP, GDP, CDP or their any combination.
The mixture comprising D-fluorescein and L-fluorescein keeps stable in long period section, wherein passes through light output measurement stability when mixture is used in uciferase activity mensuration.The stability of described increase can be the signal stabilization of storage time and/or the increase increased.(store after 8 hours or after storing 4 days at 4 DEG C, it can show the loss of activity of about 10% at 22 DEG C) compared with the preparation of routine, the stability that preparation display of the present invention increases, namely increase storage time/stability.When such as about 4 DEG C (such as about 1 DEG C to about 6 DEG C) or about 20 DEG C to 25 DEG C or about 20 DEG C to about 35 DEG C, such as about 22 DEG C (such as about 20 DEG C to about 25 DEG C, about 20 DEG C to about 35 DEG C, about 22 DEG C to about 35 DEG C, about 22 DEG C to about 30 DEG C, about 25 DEG C to about 35 DEG C, or about 25 DEG C to about 30 DEG C) temperature under when storing, in storage at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, after 4 years or longer time period, detect at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% original activity (wherein, uciferase activity survey be fixed at T=0 under a given set condition, measure original activity and under substantially the same condition in the activity subsequently of point in time measurement afterwards).Such as, at about 20 DEG C to about 25 DEG C, about 20 DEG C to about 35 DEG C, about 22 DEG C to about 35 DEG C, about 22 DEG C to about 30 DEG C, about 25 DEG C to about 35 DEG C, at the temperature of about 25 DEG C to about 30 DEG C, about or at least about after 2 months hatch, use the remaining original activity that the method as herein described of the L-fluorescein of about 50:50 and the mixture of D-fluorescein, composition and test kit will have at least about 50%.
Uciferase activity measures and comprises, such as, and the luciferase in the damping fluid of the pH of the most applicable used enzyme, fluorescein (such as 1mM), ATP (such as, 3mM), MgSO 4(such as, 15mM).Other optional component can be there is to optimize or to stablize the activity of luciferase used.The example that uciferase activity measures is described in this paper embodiment.In some embodiments, luciferase can be purified, or can in sample such as reporter gene expressing luciferase.
Provide the test kit of the mixture comprising D-fluorescein and L-fluorescein, method and composition in this article.Described test kit, method and composition can comprise one or more luciferases, or may be used for using luciferase reporter gene to be determined in the prokaryotic cell prokaryocyte of conversion or the eukaryotic cell of transfection and to measure luciferase.Described cell can be, such as, bacterial cell, as kind (Streptomycesspp.), the kind (Bacillusspp.) of bacillus, the kind (Staphylococcusspp.) etc. of Staphylococcus of intestinal bacteria (E.coli), streptomyces; Mammiferous cell, as ox, goat, sheep, dog, cat, non-human primate (as ape), and people's cell; Mammal cell line, as CHO, COS, HEK293, HeLa, CV-1, SH-SY5Y and NIH3T3 cell; Vegetable cell (dicotyledonous or unifacial leaf) or fungal cell, as yeast, such as, Pichia (Pichia), yeast belong (Saccharomyces) or Schizosaccharomyces (Schizosaccharomyces).
Utilize fluorescein as the luciferase of substrate, comprise beetle luciferase, such as common Lampyridea (Lampyridea section) luciferase.Beetle luciferase is commonly referred to Photinus pyralis LUC in the literature; But Photinus pyralis LUC is actually the subgroup of beetle luciferase class.Beetle luciferase also comprises Pleonomus luciferase, as from Jamaica Pleonomus (Pyrophorusplagiophthalamus).Beetle luciferase can by the lantern purification of beetle itself or by protein expression system purifying as known in the art.
Beetle luciferase, the Photinus pyralis LUC particularly from North America Lampyridea (Photinuspyralis) or Pennsylvania Lampyridea (Photurispennsylvanica) is well known in the art.The luciferase (LucPpy) of North America fluorescence worm is by such as consisting of about 550 amino acid (61kDa) calculated by the nucleotide sequence coded protein of gene.Pennsylvania Lampyridea (Photurispennsylvanica) Photinus pyralis LUC (LucPpe2) forms (GenBank2190534 by 545 amino-acid residues; The people such as Ye, 1997).The sudden change luciferase (such as thermostability and/or chemical stability) being derived from LucPpe2 can comprise LucPpe2m78 (also referred to as 78-0B10), LucPpe2m90 (also referred to as 90-1B5), LucPpe2ml33 (also referred to as 133-1B2) and LucPpe2ml46 (also referred to as 146-1H2).But the luciferase of the listed restriction of any satisfied this paper may be used for composition of the present invention, method and test kit.PCT/US99/30925 discloses and obtains thermally-stabilised and/or chemically stable luciferase as the method for LucPpe2m78, LucPpe2m90, LucPpe2ml33 and LucPpe2ml46.
The luciferase of separation and/or purifying can be used.The contaminant component of luciferase natural surroundings usually to disturb the material of luciferase assay, and can comprise the material of enzyme, hormone and other protein properties or non-proteinaceous.A kind of determine the technology of purity be use under being applied in non-reduced or reductive condition Coomassie blue or silver dye SDS-PAGE analyze.
Luciferase for composition of the present invention, test kit and method comprises those luciferases producing stable signal and/or have thermostability and/or chemical stability, that is, they produce the lighting time interval (luminous intensity be defined as when starting relative to luciferase reaction is less than the luminous intensity loss of 50% for every 30 minutes) of increase or produce larger enzyme stability at a higher temperature in luciferase reaction.Exemplary fluorescence element enzyme in U.S. Patent No. 6,132,983,6,171,808,6,265,177,6,602,677,7,241,584,7,906,298 and 8,030,017 and U.S. Patent Application Publication No.2003-0068801, open in 2006-0183212,2009-0137019,2011-0177540 and 2012-0009647, its respective disclosure is incorporated herein by reference in their entirety at this.
Luciferase in composition of the present invention, test kit and method also comprises generation " flash of light " signal and the transformation period of light output is less than about 30 minutes, is less than about 25 minutes, is less than about 20 minutes, is less than about 15 minutes, is less than about 10 minutes, is less than about 5 minutes, is less than about 4 minutes, is less than about 3 minutes, is less than about 2 minutes or is less than the luciferase of about 1 minute.
Luciferase is included in the thermostability at least 2 hours showing increase at 50 DEG C, or at 50 DEG C those of at least 5 hours.Thermostability luciferase, when being dissolved in the suitable aqueous solution, comprise the stable transformation period for be greater than about 2 hours at about 50 DEG C, about 5 hours are greater than at 50 DEG C, be greater than about 10 hours at 50 DEG C, be greater than about 5 hours at about 60 DEG C, be greater than about 10 hours at about 60 DEG C, be greater than about 24 hours at about 60 DEG C, be greater than about 3 months or be greater than the luciferase of about 6 months at about 22 DEG C at about 22 DEG C.
In certain embodiments, as described herein, the amount being present in the luciferase in the reaction mixture of the racemic mixture comprising L-fluorescein and D-fluorescein can increase to provide the light output of the similar reaction mixture being substantially equal to the racemic mixture not comprising L-fluorescein and D-fluorescein.Such as, the amount of luciferase can be increased to the fluorescein enzyme amount in the reaction mixture of the racemic mixture not comprising L-fluorescein and D-fluorescein at least about 120%, at least about 150%, at least about 200% (2 times), at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500% (5 times), at least about 600%, at least about 700%, at least about 800%, at least about 900% or at least about 1000% (10 times), to realize the light output of equivalence substantially.
In certain embodiments, composition as herein described, method and test kit provide the transformation period of the increase of the luminous signal produced during luciferase reaction, that is, the signal stabilization of increase.With by comprising substantially pure D-fluorescein, or comprise D-fluorescein and be substantially free of L-fluorescein, the luminous signal that the luciferase reaction such as comprising micro-L-fluorescein produces is compared, the composition containing the mixture of D-fluorescein and L-fluorescein as herein described, method and test kit can increase the transformation period (luminous intensity produced in luciferase reaction) of luminous signal at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450% or at least about 500%.In some embodiments, with by comprising substantially pure D-fluorescein or comprising D-fluorescein and be substantially free of L-fluorescein, the luminous signal that the luciferase reaction such as comprising micro-L-fluorescein produces is compared, achieve the luminous signal transformation period increase at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12 times, at least about 15 times or at least about 20 times.
Light output can be measured by relative light unit (RLU).In some embodiments, when using the mixture of D-fluorescein and L-fluorescein, the initial light output of fluorescent reaction is lower.Such as, with comprise substantially pure D-fluorescein, or comprise D-fluorescein and be substantially free of L-fluorescein, the luciferase reaction such as comprising micro-L-fluorescein is compared, what described initial light output can be initial light output is less than about 95%, be less than about 90%, be less than about 85%, be less than about 80%, be less than about 75%, be less than about 70%, be less than about 65%, be less than about 60%, be less than about 55%, be less than about 50%, be less than about 45%, be less than about 40%, be less than about 35%, be less than about 30%, be less than about 25%, be less than about 20%, be less than about 15%, be less than about 10% or be less than about 5%.In some embodiments, as described herein, lower initial light output is with the increase of the transformation period of fluorescent reaction.
In test kit as herein described, method and composition, luciferase can provide with solution such as the aqueous solution containing L-fluorescein and D-fluorescein, or can with the form (it dissolves before use) of powdery or drying, such as, provide in the container be separated with L-fluorescein with D-fluorescein.In some embodiments, luciferase provides in the container be separated with L-fluorescein with D-fluorescein with solubilized form.Be included in composition as herein described, no matter fluorescein in test kit and method (is D-fluorescein, the combination of L-fluorescein or D-fluorescein and L-fluorescein) amount can be at least about 1nM, at least about 5nM, at least about 10nM, at least about 50nM, at least about 100nM, at least about 0.5 μM, at least about 1 μM, at least about 5 μMs, at least about 10 μMs, at least about 25 μMs, at least about 50 μMs, at least about 100 μMs, at least about 250 μMs, at least about 0.5mM, about 50mM is less than at least about 1mM, be less than about 40mM, be less than about 30mM, be less than about 30mM, be less than about 10mM, be less than about 5mM, be less than about 4mM, be less than about 3mM or be less than about 2mM.
Except the mixture of D-fluorescein as herein described and L-fluorescein, test kit as herein described, composition and method can comprise one or more additional component, as luciferase; Buffer reagent is as citric acid or citrate buffer agent, MES, Isosorbide-5-Nitrae-piperazine two ethyl sulfonic acid or HEPES; Inorganic phosphate, such as tetra-sodium or potassiumphosphate form; Sequestrant is as EDTA, CDTA or 1,2-diamino-cyclohexane tetraacethyl; Salt, as Sodium Fluoride, magnesium sulfate; Tensio-active agent or washing composition as (such as non-ionic type nonyl phenol ethoxylate), Trimethyllaurylammonium bromide (DTAB) or (hydroxyl polyethoxye dodecane); Defoamer as dF204 (organic defoamer) or dF (silicone defoamer); Protein stabilizing agent as gelatin, 10% (gelatin, A type) or albumin (such as BSA, HSA) or glycerine; Adenosine triphosphate (ATP) or adenosine monophosphate (AMP).Other component can comprise polyoxyethylene glycol, polyvinyl pyridine, crown ether or cyclodextrin.Additional component can comprise, and such as, following one or more: mercaptan compound, as coenzyme A; Reductive agent, as dithiothreitol (DTT) (DTT), as the sulfocompound of reductive agent, as sulphite, thiosulphate.
The washing composition that can be included in test kit, composition and method comprises cationic detergent, anionic detergent, nonionic detergent or zwitterionic detergent.Washing composition can comprise, such as, washing composition (polyglycol ether (non-ionic type)), washing composition (polyoxyethylene 23 lauryl ether), washing composition (polyoxyethylene 20 cetyl ether (HO (CH 2cH 2o) 20c 16h 33)), Triton washing composition (4-(1,3,3-tetramethyl butyl) phenyl-polyoxyethylene glycol (t-Oct-C 6h 4-(OCH 2cH 2) xoH, x=9-10)), Triton washing composition (4)-(1,1,3,3-tetramethyl butyl) phenyl-polyoxyethylene glycol), Triton washing composition (polyoxyethylene-9,10 branched nonyl phenyl ether), washing composition (3-[(3-courage amidopropyl) diformazan ammonium]-1-propanesulfonic acid salt), washing composition (3-[3-courage amidopropyl] dimethylammonio-2-hydroxyl-1-propanesulfonic acid salt), washing composition (N, N-bis-(3-D-glucose cocamidopropyl) courage acid amides), stain remover (poly(oxyethylene glycol) 400 lauryl ether (HO (CH 2cH 2o) n(CH 2) 11cH 3)), Pluronic washing composition (PEG-block-poly-(propylene glycol)-block-PEG), Rhodasurf washing composition (polyethoxye (20) oleyl alcohol), Chemal washing composition (polyoxyethylene 9 lauryl alcohol), Sulfonyl washing composition (2,4,7,9-tetramethyl--5-decine (deceyne)-4,7-diol ethoxylate 10), deoxycholate salt, CTA3, Pierce washing composition (C8=octyl group-β-D-glucopyranoside) or Pierce washing composition (the positive decyl of n--β-D-Maltose glycosides (C10 alkyl group side chain)).
In method as herein described, the mixture containing D-fluorescein and L-fluorescein used in test kit and composition or in the mensuration of the D-of utilization fluorescein as herein described and L-fluorescein can have at least about 5, at least about 5.1, at least about 5.2, at least about 5.3, at least about 5.4, at least about 5.5, at least about 5.6, at least about 5.7, be at least about 5.8, at least about 5.9, at least about 6, at least about 6.1, at least about 6.2, at least about 6.3, at least about 6.4, at least about 6.5, at least about 6.6, at least about 6.7, at least about 6.8, at least about 6.9, at least about 7, at least about 7.1, at least about 7.2, at least about 7.3, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, at least about 8, at least about 8.1, at least about 8.2, be at least about 8.3, at least about 8.4, or be less than about 9 at least about 8.5, be less than about 8.9, be less than about 8.8, be less than about 8.7, be less than about 8.6, be less than about 8.5, be less than about 8.4, be less than about 8.3, be less than about 8.2, be less than about 8.1, be less than about 8, be less than about 7.9, be less than about 7.8, be less than about 7.7, be less than about 7.6, be less than about 7.5, be less than about 7.4, be less than about 7.3, be less than about 7.2, be less than about 7.1, be less than about 7, be less than about 6.9, be less than about 6.8, be less than about 6.7, be less than about 6.6, or be less than the pH of about 6.5.At blend composition or measure in thing certain pH can be kept to provide the environment promoting luminescence bright fast, the pH of 8 to 9 according to appointment, or slow luminous thus provide more lasting fluorescence, as be less than about 7.5 or be less than about 7 pH.
Before storage certain hour section, D-fluorescein and L-fluorescein can combine with arbitrary ratio as herein described.Before storage, the luciferase of test kit as herein described or composition, ATP or any other component can optionally merge with the mixture of D-fluorescein and L-fluorescein.This mixture can at least about-85 DEG C, at least about-80 DEG C, at least about-75 DEG C, at least about-50 DEG C, at least about-40 DEG C, at least about-30 DEG C, at least about-25 DEG C, at least about-20 DEG C, at least about 0 DEG C, at least about 1 DEG C, at least about 2 DEG C, at least about 3 DEG C or be less than about 45 DEG C at least about 4 DEG C, be less than about 40 DEG C, be less than about 38 DEG C, be less than about 35 DEG C, be less than about 30 DEG C, be less than about 25 DEG C, be less than about 22 DEG C, be less than about 21 DEG C, be less than about 20 DEG C, be less than about 15 DEG C, be less than about 10 DEG C, be less than about 8 DEG C or be less than about 6 DEG C temperature under store certain hour section.The time period stored can be at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 1 day, at least about 1 week, at least about 2 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, or at least about 12 months.After between the shelf lives amount of remaining D-fluorescein can be store before in mixture initial D-fluorescein amount at least about 99%, at least about 98%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75% or at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 54%, at least about 53%, at least about 52% or at least about 51%.After between the shelf lives amount of remaining D-fluorescein can be store before in mixture D-fluorescein original bulk at least about 101%, at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 750% or at least about 1000%, such as, if described preparation comprised the L-fluorescein more than D-fluorescein before storage.
By producing luminous luciferase-luciferin reaction, composition as herein described and test kit may be used for existence or the amount of ATP in working sample.Utilizing the reaction of ATP, such as, after kinase reaction, in sample, can ATP there is, with the amount making the ATP in sample and the luminous intensity that produces with utilize the amount of the activity of the enzyme of ATP to be inversely proportional to.Sample can containing complete cell, and can be crude or the cell pyrolysis liquid of clarification or can comprise purified enzyme, described enzyme produces or uses ATP.Described cell can be prokaryotic cell prokaryocyte or eukaryotic cell.
Composition disclosed herein and test kit may be used for existence and the amount of luciferase in working sample.At transfection such as luciferase gene, such as, after the instantaneous or stable transfection of reporter gene, in sample, luciferase can be there is.Described sample can containing complete cell, the cell pyrolysis liquid of unprocessed or clarification or comprise whole animal, such as, to whole animal imaging.In cell-free extract after expressing luciferase encoding sequence such as rabbit reticulocyte lysate or wheatgerm translation system, in sample, luciferase can be there is.Described cell can be prokaryotic cell prokaryocyte or eukaryotic cell.
But to should be appreciated that in its application the present invention is not limited to the listed or CONSTRUCTED SPECIFICATION shown in the following drawings and component arrangement in the de-scription.The present invention can implement other embodiment, and can implement in every way or carry out.
Should be appreciated that any numerical range described in the description comprises all values from lower value to higher limit.Such as, if concentration range is described as 1% to 50%, be then intended that, in this explanation, clearly illustrate the value of such as 2% to 40%, 10% to 30% or 1% to 3% etc.Should be appreciated that any numerical range quoted from the description comprises all values from least described lower value (not containing the upper limit), and height is to all values of described higher limit (not containing lower limit).These are only the examples of concrete intention, and between the minimum value enumerated and maximum value and all possible combinations of values comprising described minimum value and maximum value be regarded as clearly stating in this application.
But should be appreciated that the present invention is not limited to CONSTRUCTED SPECIFICATION listed in the de-scription and component arrangement in its application.In addition, should be appreciated that the wording that uses in this specification sheets and term are the objects in order to describe, and should not be regarded as limiting object.In description context of the present invention, term " " and " one " and " described " and similar indicator will be interpreted as containing odd number and plural form, unless be otherwise noted in this specification sheets or obvious contradiction in context.Term " comprises ", " having ", " comprising " and " containing " will be interpreted as open-ended term (that is, meaning " including but not limited to "), except as otherwise noted.Unless be otherwise noted in this manual or separately had obvious contradiction in context, otherwise all methods described in carry out this can specification sheets with any suitable order.The patent application of quoting from herein, patent and citing document clearly and pass completely through introduce entirety be incorporated to.When inconsistent explanation is possible, be as the criterion with present disclosure.
Purposes only meant for illustration content of the present disclosure and the embodiment of any and all embodiments provided herein or exemplary language (such as, " as "), and do not limit the scope of claim.
Embodiment 1: the stability of acceleration
5mML-fluorescein is dissolved in CellTiter-Glo (the ATP detection reagent containing Ultra-Glo luciferase and 5mMD-fluorescein; PromegaCorporation).Aliquots containig is placed at different temperatures the time of different lengths, be then stored in-80 DEG C.All samples thaw to room temperature simultaneously, and the ATP then with 1 μM in water mixes with 1:1.Luminous intensity (RLU) is measured after 10 minutes.Result is in shown in Fig. 1.
The rate of disintegration from comparatively high temps, the rate of disintegration under using Arrhenius equation to calculate lesser temps.At 22 DEG C, calculate 10% performance loss and occurred 8.6 days time.At 4 DEG C, calculate 10% and 50% loss and occur when 4.2 months and 2 years respectively.At-20 DEG C, calculate 10% be lost in 20 years time occur.
Embodiment 2: at the real-time stability of 22 DEG C.
By there is and do not have the aliquots containig of the CellTiter-Glo of 5mML-fluorescein in the time of 22 DEG C of placement different lengthss, be then stored in-80 DEG C.All samples thaw to room temperature simultaneously, and the ATP then with 1 μM in water mixes with 1:1.Luminous intensity (RLU) is measured after 10 minutes.Result is in shown in Fig. 2.
For the CellTiter-Glo and have without L-fluorescein, at 22 DEG C, 10% performance variation occurs when about 12 hours or about 2 weeks respectively.
The comparison of embodiment 3-fluorescein and 5 '-fluorine fluorescein
5mML-isomer fluorescein or fluorine fluorescein are dissolved in the reagent (500mMMES comprising 5mM D-isomer separately, pH6,400mMKC1,3mMCDTA, 20mMMgSO4,2mMNaF, 15 μMs of tetrasodium pyrophosphate, 2%Thesit, 1%DTAB, 0.2%Mazu and 0.21mg/mlUltra-Glo) in.Then, the often kind of reagent comprising CellTiter-Glo mixes with 1:1 with 2 μMs of ATP in water, and measures the luminous intensity (RLU) along with time lapse.Result is in shown in table 1.
Table 1
Although only there is moderate differences when comparing D-isomer in light output, the isomer mixture of 5 '-fluorine fluorescein is significantly brighter than the isomer mixture of the fluorescein with the roughly the same signal transformation period.
The stability of the increase of the reagent of embodiment 4. containing natural Photinus pyralis LUC
1mML-fluorescein is dissolved in the reagent (50mM magnesium acetate, the 25mMTris acetic acid that comprise 1mMD-fluorescein and the natural Photinus pyralis LUC of 13.54 μ g/ml, pH7.75,0.1mMEDTA, 0.002% trinitride, 0.5mMDTT, 1.3mg/mlBSA) ( restructuring luciferase, Promega, MadisonWI).Aliquots containig is placed at 22 DEG C the time of different lengths, be then stored in-80 DEG C.All samples thaw to room temperature simultaneously, are then mixed with 1:1 with the ATP of 20nM in water by injection.After within 10 seconds, postponing, measure luminous intensity (RLU) and quadrature within 10 seconds.Result is in shown in table 3.
For the reagent or have without L-fluorescein, at 22 DEG C, the performance variation of 10% littlely to occur constantly about 5 hours or about 50 respectively.
Embodiment 5. luciferase reporter gene measures D-/L-fluorescein ratios different in reagent to luminous intensity and the impact of signal transformation period
By the 2.5x10 of the HEK293 cell of expressing luciferase stably (CMV-Fluc) in 100 μ l perfect mediums (DMEM+10% foetal calf serum+1 × NEAA (non-essential amino acid)) 5individual cell/ml is plated in white 96 orifice plates.Then by described cell at 37 DEG C, 5%CO 2lower overnight incubation.After night incubation, flat board is made to equilibrate to 22 DEG C.Add the 100 μ l Photinus pyralis LUC reporter-gene assays reagent (wherein D-5 '-fluorine fluorescein/L-5 '-fluorine fluorescein is 100:0,95:5,90:10,85:15,80:20 or 75:25) containing 1mM fluorescein in cell (n=3), and under agitation hatch 3 minutes.? multi+ is upper measures luminous intensity in every 3 minutes at 22 DEG C, continues 5 hours.
Fig. 4 shows the impact of different D-/L-fluorescein ratios on initial luminance (RLU) (A) and signal transformation period (B).By luminous intensity and single exponent ring-down matching are calculated the signal transformation period.
The D-/L-fluorescein ratio that embodiment 6. is different measures the impact of the stability of reagent on luciferase reporter gene
Preparation has the Photinus pyralis LUC reporter-gene assays reagent of the D-/L-5 '-fluorine fluorescein of different ratio (100:0,90:10,80:20,70:30,60:40 and 50:50).Often kind of reagent be divided into 180 μ l aliquots containigs and in 22 DEG C of water-baths, hatch different time amount (0,1,2,3,4 or 5 day).After each time period, by freezing at-80 DEG C for the aliquots containig of the reagent of each ratio.After time course terminates, all aliquots containigs are thawed, make mixing by inhaling up and down, and 50 μ l are transferred in the triplicate hole of 96 orifice plates, then 22 DEG C of balances.In the DMEM being supplemented with 0.1%Prionex, the firefly luciferin (QuantiLum) of purifying be diluted to 0.69 μ g/ml and add 50 μ l to every hole, and mixing 3 minutes.? multi+ photometer reads luminous intensity.
The ratio that Fig. 5 shows when D-/L-5 ' fluorine fluorescein increases close to the room temperature stability of luciferase reporter gene mensuration reagent during 50:50.

Claims (100)

1. a composition, it comprises luciferase, L-fluorescein and D-fluorescein, and wherein said composition is substantially free of ATP.
2. a composition, it comprises D-fluorescein, L-fluorescein and ATP, and wherein said composition is substantially free of luciferase
3. a composition, it comprises D-fluorescein, L-fluorescein and ATP, and the concentration of wherein said L-fluorescein exceedes the concentration of described D-fluorescein.
4. composition according to claim 1 and 2, wherein said composition comprises at least about 0.5:1 to the L-fluorescein of about 2:1 and the ratio of D-fluorescein.
5. composition according to claim 1 and 2, wherein said composition comprises at least about 5:95 to the L-fluorescein of about 55:45 and the ratio of D-fluorescein.
6. composition according to any one of claim 1 to 5, wherein said composition has at least about 5 and is less than the pH of about 9.
7. composition according to any one of claim 1 to 6, wherein said luciferase is beetle luciferase, as Photinus pyralis LUC.
8. composition according to any one of claim 1 to 7, wherein said D-fluorescein is D-5 ' fluorine fluorescein, and described L-fluorescein is L-5' fluorine fluorescein.
9. composition according to any one of claim 1 to 8, wherein said composition also comprises at least one and is selected from following additional component: buffer reagent, protein stabilizing agent, magnesium ion, metal chelator, washing composition, defoamer, inorganic phosphate, pyrophosphate salt, AMP, coenzyme A, reductive agent or its combination.
10. composition according to claim 9, wherein said composition comprises magnesium sulfate and anti-form-1,2-cyclohexanediaminetetraacetic acid (CDTA).
11. compositions according to any one of claim 1 to 8, wherein said composition also comprises at least one and is selected from following additional component: 1,4-piperazine two ethyl sulfonic acid, 1,2-diamino-cyclohexane tetraacethyl, Tergitol washing composition, Mazu defoamer, magnesium sulfate, ATP, coenzyme A, pyrophosphate salt, DTT, sulphite and thiosulphate.
12. compositions according to any one of claim 1 to 8, wherein said composition also comprises at least one and is selected from following component: Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate, ethylenediamine tetraacetic acid (EDTA) (EDTA) and AMP.
13. compositions according to claim 9,11 or 12, wherein said composition comprises at least two kinds in described additional component.
14. compositions according to claim 9,11 or 12, wherein said composition comprises at least three kinds in described additional component.
15. compositions according to claim 9,11 or 12, wherein said composition comprises at least four kinds in described additional component.
16. compositions according to claim 9,11 or 12, wherein said composition comprises at least five kinds in described additional component.
17. compositions according to any one of claim 1 to 8, wherein said composition also comprises magnesium ion, citric acid buffer agent, MES buffer reagent, Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate and ethylenediamine tetraacetic acid (EDTA) (EDTA).
18. compositions according to any one of claim 1 to 8, wherein said composition comprises citric acid buffer agent, HEPES buffer reagent, magnesium ion, ethylenediamine tetraacetic acid (EDTA) (EDTA), potassiumphosphate, defoamer, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane and AMP.
19. compositions according to claim 7, wherein said beetle luciferase is Photinus pyralis LUC or Pleonomus luciferase.
20. compositions according to aforementioned any one claim, wherein compared with substantially not having the composition of L-fluorescein, when storing at the temperature of about 0 DEG C to about 35 DEG C, described composition has storage time and/or the stability of increase.
21. 1 kinds of compositions, it has storage time and/or the stability of increase, and described composition comprises luciferase, L-fluorescein and D-fluorescein, and wherein said composition is substantially free of ATP.
22. compositions according to claim 21, wherein said luciferase is beetle luciferase.
23. compositions according to claim 21 or 22, wherein said beetle luciferase is Photinus pyralis LUC or Pleonomus luciferase.
24. 1 kinds of compositions, it has storage time and/or the stability of increase, and described composition comprises D-fluorescein, L-fluorescein and ATP, and wherein said composition is substantially free of luciferase.
25. compositions according to any one of claim 21-24, wherein said composition comprises at least about 0.5:1 to the L-fluorescein of about 2:1 and the ratio of D-fluorescein.
26. compositions according to any one of claim 21-24, wherein said composition comprises at least about 5:95 to the L-fluorescein of about 55:45 and the ratio of D-fluorescein.
27. 1 kinds of compositions, it has storage time and/or the stability of increase, and described composition comprises D-fluorescein, L-fluorescein and ATP, and the concentration of wherein said L-fluorescein exceedes the concentration of described D-fluorescein.
28. compositions according to any one of claim 21 to 27, wherein compared with not there is the composition of L-fluorescein substantially, when storing at the temperature of about 0 DEG C to about 35 DEG C, described reaction mixture has storage time and/or the stability appearance of increase.
29. compositions according to any one of claim 21 to 28, wherein said composition has at least about 5 and is less than the pH of about 9.
30. compositions according to any one of claim 21 to 29, wherein said D-fluorescein is D-5 ' fluorine fluorescein, and described L-fluorescein is L-5' fluorine fluorescein.
31. compositions according to any one of claim 21 to 30, wherein said composition also comprises at least one and is selected from following additional component: buffer reagent, protein stabilizing agent, magnesium ion, metal chelator, washing composition, defoamer, inorganic phosphate, pyrophosphate salt, AMP, coenzyme A, reductive agent or its combination.
32. compositions according to claim 31, wherein said composition comprises magnesium sulfate and anti-form-1,2-cyclohexanediaminetetraacetic acid (CDTA).
33. compositions according to any one of claim 21 to 32, wherein said composition also comprises at least one and is selected from following additional component: 1,4-piperazine two ethyl sulfonic acid, 1,2-diamino-cyclohexane tetraacethyl, Tergitol washing composition, Mazu defoamer, magnesium sulfate, ATP, coenzyme A, pyrophosphate salt, DTT, sulphite and thiosulphate.
34. compositions according to any one of claim 21 to 33, wherein said composition also comprises at least one and is selected from following component: Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate, ethylenediamine tetraacetic acid (EDTA) (EDTA) and AMP.
35. compositions according to claim 31,33 or 34, wherein said composition comprises at least two kinds in described additional component.
36. compositions according to claim 31,33 or 34, wherein said composition comprises at least three kinds in described additional component.
37. compositions according to claim 31,33 or 34, wherein said composition comprises at least four kinds in described additional component.
38. compositions according to claim 31,33 or 34, wherein said composition comprises at least five kinds in described additional component.
39. compositions according to any one of claim 21 to 30, wherein said composition also comprises magnesium ion, citric acid buffer agent, MES buffer reagent, Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate and ethylenediamine tetraacetic acid (EDTA) (EDTA).
40. compositions according to any one of claim 21 to 30, wherein said composition comprises citric acid buffer agent, HEPES buffer reagent, magnesium ion, ethylenediamine tetraacetic acid (EDTA) (EDTA), potassiumphosphate, defoamer, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane and AMP.
41. 1 kinds of test kits, it comprises the composition according to any one of Claims 1-4 0 be packaged at least one container.
42. 1 kinds of test kits, it comprises and is packaged in the composition according to any one of claim 1 to 8 and 19 to 30 in the first container and second container, and described second container comprises at least one and is selected from following component: citric acid buffer agent, MES buffer reagent, Sodium Fluoride, dodecyl trimethyl ammonium, hydroxyl polyethoxye dodecane, defoamer, pyrophosphate salt, potassiumphosphate, ethylenediamine tetraacetic acid (EDTA) (EDTA) and AMP.
43. test kits according to claim 41 or 42, wherein compared with substantially not having the composition of L-fluorescein, when storing at the temperature between about 0 DEG C to about 35 DEG C, described composition has storage time and/or the stability of increase.
44. 1 kinds for the existence of ATP in working sample or the method for amount, it comprise make the composition according to any one of claim 1,4 to 17,19 to 23,25,26 and 28-39 and sample contacts and detection the luminous intensity that produces, measure existence or the amount of ATP in described sample thus.
45. methods according to claim 44, wherein said sample comprises complete cell.
46. methods according to claim 44, wherein said sample comprises the cell lysate of unprocessed cell lysate or clarification.
47. methods according to claim 45 or 46, wherein said cell is prokaryotic cell prokaryocyte or eukaryotic cell.
48. methods according to any one of claim 44 to 47, wherein said sample comprises purified enzyme, and described enzyme produces or uses ATP.
49. methods according to any one of claim 44 to 48, wherein compared with substantially not having the composition of L-fluorescein, when storing at the temperature between about 0 DEG C to about 35 DEG C, described composition has storage time and/or the stability of increase.
50. 1 kinds for detect expressing luciferase cell in the method for uciferase activity, described method is included in cells luciferase; Make described luciferase, described cell or its combination and comprise the mixture of L-fluorescein with D-fluorescein and contact and detect luminous intensity, the ratio of described L-fluorescein and described D-fluorescein is that (i) is at least about 5:95 to about 75:25, (ii) at least about 5:95 to about 50:50, (iii) at least about 5:95 to about 25:75 or (iv) at least about 5:95 to about 20:80.
51. methods according to claim 50, wherein make described lysis, and the cell of the described cracking comprising described luciferase is contacted with described mixture.
52. methods according to claim 51, wherein make the cell of described cracking clarify to form the cell extract comprising the clarification of described luciferase, and the cell extract of described clarification are contacted with described mixture.
53. methods according to any one of claim 50 to 52, wherein said cell is eukaryotic cell, as mammalian cell.
54. methods according to any one of claim 50 to 53, wherein said mixture increases the transformation period of described luminous signal.
55. methods according to any one of claim 50 to 54, wherein use cell described in luciferase-encoding sequences transient transfection.
56. methods according to any one of claim 50 to 55, wherein said mixture have at least about 6.5 to be less than about 7.8 pH or at least about 6.6 at least about 6.8 pH.
57. methods according to any one of claim 50 to 56, wherein said D-fluorescein is D-5' fluorine fluorescein, and described L-fluorescein is L-5' fluorine fluorescein.
58. methods according to any one of claim 50 to 57, wherein compared with substantially not having the composition of L-fluorescein, when storing at the temperature between about 0 DEG C to about 35 DEG C, described composition has storage time and/or the stability of increase.
59. 1 kinds for carrying out the method for luciferase reaction, described method comprises:
A time period that () will store at the temperature of the mixture comprising L-fluorescein and D-fluorescein between about 0 DEG C to about 35 DEG C at least about 4 hours, wherein when merging with sample in the mensuration thing comprising luciferase and ATP, the mixture of described storage allows to produce light output, and the described light output after the wherein said time period be described light output when starting the described time period at least about 50%; And
B () is after step (a), allow the mixture of described storage and (i) described sample and (ii) ATP or ATP source, luciferase or its combine to merge and measures thing with forming reactions, and the luminous intensity that detection produces.
60. methods according to claim 59, the wherein said time period is at least about 12 hours, and described temperature is about 0 DEG C to about 6 DEG C or about 20 DEG C to about 35 DEG C.
61. methods according to claim 59 or 60, wherein said sample comprises luciferase, and described mixture comprises ATP.
62. methods according to claim 59 or 60, wherein said sample comprises ATP, and described mixture comprises luciferase.
63. methods according to any one of claim 59 to 62, the described light output after the wherein said time period be described light output when starting the described time period at least about 75%.
64. 1 kinds for preparing the method for the component for luciferase reaction, described method comprises:
A D-fluorescein and L-fluorescein merge to form mixture by (); And
B described mixture is stored the time period at least about 2 days by () at the temperature of about 0 DEG C to about 35 DEG C,
Wherein, when merging with sample in the mensuration thing comprising luciferase, described mixture allows to produce light output, and the described light output after the wherein said time period be described light output when starting the described time period at least about 50%.
65. methods according to any one of claim 59 to 64, wherein said mixture is substantially free of ATP or luciferase.
66. methods according to any one of claim 59 to 65, wherein said temperature is about 20 DEG C to about 35 DEG C.
67. according to the method described in claim 59 to 66, and wherein said temperature is about 0 DEG C to about 6 DEG C, and the described time period is at least about 2 weeks.
68. methods according to any one of claim 59 to 67, the wherein said time period is at least about 12 weeks.
69. methods according to any one of claim 59 to 68, the wherein said time period is at least about 26 weeks.
70. methods according to any one of claim 59 to 69, the described light output after the wherein said time period be described light output when starting the described time period at least about 90%.
71. methods according to any one of claim 59 to 70, the described light output after the wherein said time period be described light output when starting the described time period at least about 50%.
72. methods according to any one of claim 64 to 71, after it is also included in step (b), by the mixture of described storage and (i) luciferase, (ii) ATP or ATP source or (iii) its combine and merge to form the second mixture, and detect the luminous intensity produced in described second mixture.
73. methods according to any one of claim 50 to 55 or 57 to 72, wherein said mixture has the pH of about 5 to about 9.
74. methods according to any one of claim 59 to 73, wherein said mixture comprises at least about 5:95 to the L-fluorescein of about 75:25 and the ratio of D-fluorescein.
75. 1 kinds of methods for increasing the transformation period of luminous signal, described method comprises:
A sample that () makes to comprise luciferase with comprise D-fluorescein, L-fluorescein and to contact with the mixture of ATP and measure thing with forming reactions; And
B () detects luminous intensity; Wherein with comprise D-fluorescein and compared with the comparable reaction assay thing of essentially no L-fluorescein, the transformation period of described luminous signal increases.
76. according to the method described in claim 75, and the transformation period of wherein said luminous signal adds at least about 25%.
77. according to the method described in claim 75, and the transformation period of wherein said luminous signal adds at least about 5 times.
78. methods according to any one of claim 75 to 77, wherein said sample comprises the cell of expressing described luciferase.
79. according to the method described in claim 78, wherein makes described lysis, and sample comprises the cell of the described cracking comprising described luciferase.
80. methods according to any one of claim 75 to 77, wherein sample comprises the cell extract of the clarification containing described luciferase.
81. methods according to any one of claim 78 to 80, wherein said cell is prokaryotic cell prokaryocyte or eukaryotic cell.
82. methods according to any one of claim 75 to 81, wherein said D-fluorescein is D-5' fluorine fluorescein, described L-fluorescein is L-5' fluorine fluorescein, and wherein said comparable reaction assay thing comprises D-5' fluorine fluorescein and essentially no L-5' fluorine fluorescein.
83. 1 kinds for carrying out the method for luciferase reaction, described method comprises:
A () makes composition merge with sample and ATP or ATP source, luciferase or its composition and measures thing with forming reactions, described composition comprises L-fluorescein and D-fluorescein; And
B () detects the luminous intensity produced,
Wherein, compared with not there is the composition of L-fluorescein substantially, if described composition stores the time period of at least 4 hours at the temperature of about 0 DEG C to about 35 DEG C, then described composition has the stability of increase, wherein when merging with described sample in the mensuration thing comprising luciferase and ATP, the mixture of described storage allows to produce light output, and the described light output after the wherein said time period be described light output when starting the described time period at least about 50%.
84. methods according to Claim 8 described in 3, the stability wherein increased comprises the storage time of increase and/or the signal stabilization of increase.
85. methods according to Claim 8 described in 3 or 84, the wherein said time period is at least about 12 hours, and described temperature is about 0 DEG C to about 6 DEG C or about 20 DEG C to about 35 DEG C.
86. methods according to Claim 8 according to any one of 3 to 85, wherein said sample comprises luciferase, and described composition comprises ATP.
87. methods according to Claim 8 described in 3 to 85, wherein said sample comprises ATP, and described composition comprises luciferase.
88. methods according to Claim 8 according to any one of 3 to 87, the described light output after the wherein said time period be described light output when starting the described time period at least about 75%.
89. 1 kinds for preparing the method for the component for luciferase reaction, described method comprises and merges D-fluorescein and L-fluorescein to form composition, wherein compared with not there is the composition of L-fluorescein substantially, if described composition stores the time period of at least 4 hours at the temperature of about 0 DEG C to about 35 DEG C, then described composition has the stability of increase, wherein when merging with described sample in the mensuration thing comprising luciferase and ATP, the composition of described storage allows to produce light output, and the described light output after the wherein said time period be described light output when starting the described time period at least about 50%.
90. methods according to Claim 8 described in 9, the stability wherein increased comprises the storage time of increase and/or the signal stabilization of increase.
91. methods according to Claim 8 according to any one of 3 to 90, wherein said composition is substantially free of ATP or luciferase.
92. methods according to Claim 8 according to any one of 3 to 91, wherein said temperature is about 20 DEG C to about 35 DEG C.
93. methods according to Claim 8 according to any one of 3 to 92, wherein said temperature is about 0 DEG C to about 6 DEG C, and the described time period is at least about 2 weeks.
94. methods according to Claim 8 according to any one of 3 to 93, the wherein said time period is at least about 12 weeks.
95. methods according to Claim 8 according to any one of 3 to 94, the wherein said time period is at least about 26 weeks.
96. methods according to Claim 8 according to any one of 3 to 95, the described light output after the wherein said time period be described light output when starting the described time period at least about 90%.
97. methods according to Claim 8 according to any one of 3 to 96, the described light output after the wherein said time period be described light output when starting the described time period at least about 50%.
98. methods according to Claim 8 according to any one of 3 to 97, after it is also included in step (b), by described composition and (i) luciferase, (ii) ATP or ATP source or (iii) its combine and merge to form mixture, and detect the luminous intensity produced in described mixture.
99. methods according to Claim 8 according to any one of 3 to 98, wherein said composition has the pH of about 5 to about 9.
100. methods according to Claim 8 according to any one of 3 to 99, wherein said composition comprises at least about 5:95 to the L-fluorescein of about 75:25 and the ratio of D-fluorescein.
CN201480010140.5A 2013-02-22 2014-02-21 Stable formulations for luminescent detection of luciferase and nucleoside phosphates Active CN105121653B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201911407470.9A CN111004835B (en) 2013-02-22 2014-02-21 Stable formulations for luminescent detection of luciferases and nucleoside phosphates
CN202311707362.XA CN117701675A (en) 2013-02-22 2014-02-21 Stable formulations for luminescent detection of luciferases and nucleoside phosphates

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US201361767875P 2013-02-22 2013-02-22
US61/767,875 2013-02-22
US201361783726P 2013-03-14 2013-03-14
US61/783,726 2013-03-14
PCT/US2014/017562 WO2014130760A1 (en) 2013-02-22 2014-02-21 Stabilized formulation for luminescent detection of luciferase and nucleoside phosphates

Related Child Applications (2)

Application Number Title Priority Date Filing Date
CN201911407470.9A Division CN111004835B (en) 2013-02-22 2014-02-21 Stable formulations for luminescent detection of luciferases and nucleoside phosphates
CN202311707362.XA Division CN117701675A (en) 2013-02-22 2014-02-21 Stable formulations for luminescent detection of luciferases and nucleoside phosphates

Publications (2)

Publication Number Publication Date
CN105121653A true CN105121653A (en) 2015-12-02
CN105121653B CN105121653B (en) 2020-01-24

Family

ID=50241539

Family Applications (3)

Application Number Title Priority Date Filing Date
CN202311707362.XA Pending CN117701675A (en) 2013-02-22 2014-02-21 Stable formulations for luminescent detection of luciferases and nucleoside phosphates
CN201480010140.5A Active CN105121653B (en) 2013-02-22 2014-02-21 Stable formulations for luminescent detection of luciferase and nucleoside phosphates
CN201911407470.9A Active CN111004835B (en) 2013-02-22 2014-02-21 Stable formulations for luminescent detection of luciferases and nucleoside phosphates

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202311707362.XA Pending CN117701675A (en) 2013-02-22 2014-02-21 Stable formulations for luminescent detection of luciferases and nucleoside phosphates

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN201911407470.9A Active CN111004835B (en) 2013-02-22 2014-02-21 Stable formulations for luminescent detection of luciferases and nucleoside phosphates

Country Status (5)

Country Link
US (3) US9487814B2 (en)
EP (3) EP3693471B1 (en)
JP (4) JP2016509835A (en)
CN (3) CN117701675A (en)
WO (1) WO2014130760A1 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2272973B1 (en) * 2005-05-31 2015-05-27 Promega Corporation Luminogenic and fluorogenic compounds and methods to detect molecules or conditions
CN117701675A (en) 2013-02-22 2024-03-15 普洛麦格公司 Stable formulations for luminescent detection of luciferases and nucleoside phosphates
WO2019028272A1 (en) 2017-08-04 2019-02-07 Promega Corporation Compositions and methods for stabilizing benzothiazole luciferin analogs
EP4034654A4 (en) * 2019-09-26 2024-03-27 Vidyasirimedhi Institute Of Science And Tech Luciferin derivatives and a method for synthesis thereof
CN112063687B (en) * 2020-09-02 2021-06-11 创芯国际生物科技(广州)有限公司 ATP detection reagent for 3D organs and preparation method and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235961A (en) * 1978-06-29 1980-11-25 Lkb-Produkter Ab Method for photometric determination of the subunit B of creatine kinase and a reagent for carrying out the method
US4246340A (en) * 1978-05-31 1981-01-20 Lkb-Producter Ab Method and reagent for bioluminiscence
WO2001020002A1 (en) * 1999-09-15 2001-03-22 Promega Corporation Thermostable luciferases from photuris pennsylvanica and pyrophorus plagiophthalamus and methods of production

Family Cites Families (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8745893B2 (en) 2011-08-10 2014-06-10 Gavrieli Brands LLC Split-sole footwear
GB9501170D0 (en) 1994-03-23 1995-03-08 Secr Defence Luciferases
GB9417593D0 (en) 1994-09-01 1994-10-19 Secr Defence Luciferase labelling method
IN186115B (en) 1995-01-20 2001-06-23 Secr Defence Brit
GB9707486D0 (en) 1997-04-11 1997-05-28 Secr Defence Enzyme assays
EP1015601B1 (en) 1997-09-19 2015-01-07 Promega Corporation Thermostable luciferases and methods of production
DE19842901A1 (en) * 1998-09-18 2000-03-23 Lembert Nicolas New method for measuring cell activation comprises incubating luciferase-containing cells with L-luciferin and measuring the bioluminescence after applying a stimulus to the cells
GB9823468D0 (en) 1998-10-28 1998-12-23 Secr Defence Novel enzyme
US7083911B2 (en) 2001-02-16 2006-08-01 Promega Corporation Method for detection of ATP
EP2218790A3 (en) 2002-09-06 2010-08-25 Promega Corporation Kits for detecting transferase enzymatic activity
CA2508072A1 (en) 2002-12-23 2004-07-15 Promega Corporation Protecting luciferase activity from luciferase enzyme interfering compounds using detergents for the accurate measurement of luciferase activity
EP1935986B1 (en) 2005-05-31 2014-04-16 Promega Corporation Luminogenic and fluorogenic compounds and methods to detect molecules or conditions
US7530909B2 (en) 2005-06-27 2009-05-12 Sop Services, Inc. Basketball return net mounting system
BRPI0722061B8 (en) 2006-12-22 2021-06-22 Croma Pharma Ges M B H use of a polymer containing thiol group and implant
US20090075309A1 (en) 2007-09-06 2009-03-19 Gambhir Sanjiv S Bisdeoxycoelenterazine derivatives, methods of use, and BRET2 systems
WO2014007846A1 (en) 2012-07-06 2014-01-09 3M Innovative Properties Company Apparatus and methods for detecting atp in a liquid sample
CN117701675A (en) 2013-02-22 2024-03-15 普洛麦格公司 Stable formulations for luminescent detection of luciferases and nucleoside phosphates

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4246340A (en) * 1978-05-31 1981-01-20 Lkb-Producter Ab Method and reagent for bioluminiscence
US4235961A (en) * 1978-06-29 1980-11-25 Lkb-Produkter Ab Method for photometric determination of the subunit B of creatine kinase and a reagent for carrying out the method
WO2001020002A1 (en) * 1999-09-15 2001-03-22 Promega Corporation Thermostable luciferases from photuris pennsylvanica and pyrophorus plagiophthalamus and methods of production

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ERIKA M.HAWKINS等: "ONE- GloTM Luciferase Assay System: New Substrate, Better Regent", 《PROMEGA》 *
HANNAN等: "Evolution of a thermostable luciferase for application in ATP assays", 《BIOLUMINESCENCE AND CHEMILUMINESCENCE》 *

Also Published As

Publication number Publication date
EP2959010B1 (en) 2020-07-29
JP2021121218A (en) 2021-08-26
EP3693471B1 (en) 2024-01-03
JP7223057B2 (en) 2023-02-15
JP2019080577A (en) 2019-05-30
US20200063186A1 (en) 2020-02-27
JP2016509835A (en) 2016-04-04
US9487814B2 (en) 2016-11-08
WO2014130760A1 (en) 2014-08-28
CN105121653B (en) 2020-01-24
JP7128134B2 (en) 2022-08-30
CN111004835B (en) 2023-12-29
US20140242627A1 (en) 2014-08-28
EP2959010A1 (en) 2015-12-30
JP2020156493A (en) 2020-10-01
US11214822B2 (en) 2022-01-04
EP4306649A2 (en) 2024-01-17
US20160326570A1 (en) 2016-11-10
US10501774B2 (en) 2019-12-10
EP4306649A3 (en) 2024-04-03
EP3693471A1 (en) 2020-08-12
CN117701675A (en) 2024-03-15
CN111004835A (en) 2020-04-14

Similar Documents

Publication Publication Date Title
US11214822B2 (en) Stabilized formulation for luminescent detection of luciferase and nucleoside phosphates
CN105002264B (en) A kind of tolal bile acid determination reagent box
CN101948906A (en) The reagent that contains protease reaction promoter and/or colorant stabilizer
DK0980263T3 (en) Combination product combining a nucleic acid with a substance that dissolves the extracellular matrix for gene therapy
HUP0001247A2 (en) Method of improving viscosity stability of aqueous compositions
Fasching et al. Mitochondrial respiration medium-MiR06
JP4723311B2 (en) Reagent for measuring creatine kinase activity
CN101889095B (en) Methods, reagents and kits for luciferase assay
JP4781742B2 (en) Reagent for measuring creatine kinase activity
JP2019537434A (en) Thermostable luciferase with improved resistance to inhibition by luciferin degradation products
WO2002004598A3 (en) Improvement of cell culture performance by inhibitors of pyruvate dehydrogenase kinase
US20040043439A1 (en) Luciferase detection reagent kits and luciferase detection method using the kit
JP6698651B2 (en) Kit containing ATP-diphosphohydrolase for detecting bacterial ATP in a sample
EP0989189A4 (en) Reagent compositions for assaying electrolytes
WO2006013837A1 (en) Luciferase luminescence method and luminescent reagent
US6645735B2 (en) Reagent for GPT assay
KR980003585A (en) Reagent Compositions, Test Specimens, and Measurement Carts
JP2000166550A (en) Stabilization of bioluminescent reagent
JPH0937795A (en) Reagent for assaying bilirubin and assay

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant